Human phosphodiesterase IV isozymes

ABSTRACT

This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase (hPDE IV) isozymes. It also relates to polypeptides encoded by such sequences. 
     This invention also relates to an assay method for detecting the presence of such novel isozymes in human cells, and to a method of identifying compounds or other substances that inhibit or modify the activity of such isozymes.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. application Ser. No. 10/165,135, filed Jun.6, 2002 now U.S. Pat. No. 6,914,133 entitled “Human Phosphodiesterase IVIsozymes” which is a division of U.S. application Ser. No. 09/717,953,filed Nov. 21, 2000 now U.S. Pat. No. 6,489,457 which is continuation ofSer. No. 08/472,600 filed Jun. 7, 1995 which has issued as U.S. Pat. No.6,323,041, which is a continuation of U.S. application Ser. No.08/432,327 filed May 1, 1995 which is abandoned, which is a continuationof U.S. application Ser. No. 08/075,450, filed Jun. 11, 1993 which isabandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel nucleic acid sequences encoding threenovel human phosphodiesterase IV (hPDE IV) isozymes.

Cyclic nucleotide phosphodiesterases (PDE's) are a family of enzymesthat catalyze the degradation of cyclic nucleotides. Cyclic nucleotides,particularly cAMP, are important intracellular second messengers, andPDEs are one cellular component that regulates their concentration. Inrecent years, five PDE enzymes (PDE I–PDE V), as well as many subtypesof these enzymes, have been defined based on substrat affinity andcofactor requirements (Beavo J A and Reifsnyder D H, Trends Pharmacol.Sci. 11:150 [1990]; Beavo J, in: Cyclic Nucleotide Phosphodiesterases:Structure, Regulation and Drug Action. Beavo J and Housley M D (Eds.).Wiley: Chichester, pp. 3–15 [1990]).

Theophylline, a general PDE inhibitor, has been widely used in thetreatment of asthma. It has been speculated that selective inhibitors ofPDE isozymes and their subtypes (particularly the cAMP-specific PDE IV)will lead to more effective therapy with fewer side effects (forreviews, see Wieshaar RE et al., J. Med. Chem., 28:537 [1985] andGiembycz M A, Biochem. Pharm., 43:2041 [1992], Lowe J A and Cheng J B,Drugs of the Future, 17:799–807 [1992]). However, even PDE IV selectivedrugs such as rolipram suffer from emetic side effects that limit theiruse. An even more selective approach is to inhibit individual subtypesof PDE IV, each one of which is expected to have its own tissuedistribution. If the PDE IV isozyme responsible for efficacy isdifferent from that causing side effects, an isozyme selective drugcould separat therapeutic and side effects. The cloning and expressionof the human PDE IVs would greatly aid the discovery ofisozyme-selective inhibitors by providing purified isoenzymes toincorporate into drug assays.

Mammalian PDE IV, the homologue of the Drosophila Dunce gene (Chen C Net al., Proc. Nat. Acad. Sci. (USA) 83:9313 [1986]), is known to havefour isoforms in the rat (Swinnen J V et al., Proc. Nat. Acad. Sci.(USA) 86:5325 [1989]). The cloning of one human isoform of PDE IV frommonocytes was reported in 1990 (Uvi GP et al., Mol. Cell. Bio., 10:2678[1990]). From Southern blot data, the authors concluded that this enzymewas probably the only PDE IV gene in humans, with the possible exceptionof one other isozyme. The same group has recently published the sequenceof a second human isoform isolated from brain that they designate hPDEIV-B to distinguish it from the monocyte form, which they designate ashPDE IV-A (McLaughlin MM et al., J. Biol. Chem. 268:6470 [1993]). Forclarity, we will use this nomenclature as well.

Our invention relates to the nucleic acid sequences encoding three novelhuman PDE IV isozymes generated by differential splicing from a singlegene. We designate these isoforms as hPDE IV-B1, hPDE IV-B2 and hPDEIV-B3. The hPDE IV-B2 sequence encodes a polypeptide nearly identical tothat reported for hPDE IV-B (McLaughlin MM et al., J. Biol. Chem.268:6470 [1993]), and the hPOE IV-B2 splice variant represents theunspliced genomic sequence with respect to the differential splice site.Of the two other splice variants, hPDE IV-B1 encodes the longestpolypeptide chain, as well as the N-terminal sequence homologous to itsrat homologue, DPD (Colicelli J, et al., Proc. Nat. Acad. Sci. (USA)86:3599 [1989]).

The novel human PDE IV DNA sequences and their encoded peptides may beused to screen for drugs that are selective for a particular human PDEIV isozyme. Such novel DNA sequences may also be used in assays todetect the presence of a particular POE IV isozyme in human cell lines,thus providing information regarding th tissue distribution of eachisozyme and its biological relevance with respect to particular diseasestates.

The following abbreviations are used throughout this patent:

BAL bronchoalveolar lavage bp base pair(s) cAMP cyclic adenosine3′,5′-monophosphate dNTP 2′-deoxynucleoside-5′-triphosphate dATP2′-deoxyadenosine-5′-triphosphate dCTP 2′-deoxycytidine-5′-triphosphatedGTP 2′-deoxyguanidine-5′-triphosphate dTTP2′-deoxythymidine-5′-triphosphate hPDE IV-A human monocyte PDE IV hPDEIV-B human brain PDE IV hPDE IV-B1 human brain PDE IV, splice variant 1hPDE IV-B2 human brain PDE IV, splice variant 2 hPDE IV-B3 human brainPDE IV, splice variant 3 kb kilobase(s) PCR polymerase chain reactionPDE cyclic nucleotide phosphodiesterase PDE I Ca²⁺/Calmodulin-dependentPDE PDE II cGMP stimulated PDE PDE III cGMP inhibited PDE PDE IV highaffinity cAMP-specific PDE PDE V cGMP specific PDE RACE RapidAmplification of cDNA Ends RT avian myeloblastosis virus (AMV) reversetranscriptase RT-PCR PCR of RT-transcribed mRNA SSC 1X SSC = 0.15 MNaCl, 0.015 Na₃ citrate pH 7.0

The nucleotides and amino acids represented in the various sequencescontained herein have their usual single letter designations usedroutinely in the art.

SUMMARY OF THE INVENTION

This invention relates to novel nucleic acid sequences encoding thenovel hPDE IV isozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. Morespecifically, it relates to DNA segments comprising, respectively, theDNA sequences of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 and SEQUENCE IDNO. 3, as defined below, or alleleic variations of such sequences. Italso relates to polypeptides produced by expression in a host cell intowhich has been incorporated one of the foregoing DNA sequences or analleleic variation of such sequence.

This invention also relates to an isolated polypeptide comprising theamino acid sequence of SEQUENCE ID NO. 4, SEQUENCE ID NO. 5 or SEQUENCEID NO. 6.

This invention also relates to recombinant DNA comprising the DNAsequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3,or an alleleic variations of such sequence.

This invention also relates to an isolated DNA segment comprising thegenomic promoter region that regulates transcription or translation ofthe DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE IDNO. 3, or an alleleic variation of such sequence.

This invention also relates to an assay method for detecting thepresence of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3 in human cells,comprising: (a) performing a reverse transcriptase-polymerase chainreaction on total RNA from such cells using a pair of polymerase chainreaction primers that are specific for, respectively, hPDE IV-B1, hPDEIV-B2 or hPDE IV-B3, as determined from, respectively: (i) the DNAsequence of SEQUENCE ID NO. 1 or an alleleic variation thereof; (ii) thDNA sequence of SEQUENCE ID NO. 2 or an alleleic variation thereof; or(iii) the DNA sequence of SEQUENCE ID NO. 3 or an alleleic variationthereof; and (b) assaying the appearance of an appropriately sized PCRfragment by agarose gel electrophoresis.

This invention also relates to a method of identifying compounds orother substances that inhibit or modify the activity of hPDE IV-B1, hPDEIV-B2 or hPDE IV-B3, comprising measuring the activity of, respectively,hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, in: (a) a cell line into which hasbeen incorporated recombinant DNA comprising the DNA sequence ofSEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or analleleic variation thereof, or (b) a cell line that naturallyselectively expresses hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, asdetermined by the assay method described above.

This invention also relates to an isolated DNA segment comprising a DNAsequence that is a subset of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 orSEQUENCE ID NO. 3, or an alleleic variation thereof, and that is capableof hybridizing to, respectively, SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 orSEQUENCE ID NO. 3, or an alleleic variation thereof, when used as aprobe, or of amplifying all or part of such sequence when used as apolymerase chain reaction primer.

As used herein, the term “functionally equivalent DNA segment” refers toa DNA segment that encodes a polypeptide having an activity that issubstantially the same as the activity of the polypeptide encoded by theDNA to which such segment is said to be functionally equivalent.

As used herein, the term “subset of a DNA sequence” refers to anucleotide sequence that is contained in and represents part, but notall of such DNA sequence, and is sufficient to render it specific tosuch sequence when used as a PCR primer and to render it capable ofhybridizing to such sequence when used as a probe at high stringency.

The term “functionally equivalent polypeptide” refers to a polypeptidethat has substantially the same activity as the polypeptide to which itis said to be functionally equivalent.

The term “subset of a polypeptide” refers to a peptide sequence that iscontained in and represents part, but not all of such polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 Restriction MaD and CloneDiagram. This figure shows the relationship between the cDNA sequencesencoding the three splice variants. Black boxes indicate protein codingregions and open boxes indicate untranslated regions.

FIG. 2. hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 DNA and Translated AminoAcid Sequences. (+) Numbering begins with the “A” of the ATG start codonin hPDE IV-B3. Four stop codons are designated by “***”. These includethe protein translation stop (1,552), and the stop codons that preventthe coding region from coritinuing further in the 5′ direction in eachsplice variant: hPDE IV-B1 (−630), hPDE IV-B2 (−270) and hPDE IV-B3(−89). The alternate splice junction is between nucleotides −23 and −24,and the putative splice acceptor sequence in hPDE IV-B2 (−33 to −24) isunderlined.

FIG. 3. Alternative Splice Junction. This figure is a close-up view ofthe splice junction between −24 and −23, showing the three alignedsequences hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The putative spliceacceptor sequence in hPDE IV-B2 (−33 to −24) is underlined.

FIG. 4. Amino Acid Sequence Comparison: hPDE IV-B1, hPDE IV-B2, hPDEIV-B3, and Rat DPD. Identity with the hPDE IV-B1 sequence is indicatedwith a dash. A translation of the region upstream of the hPDE IV-B3start codon is shown in parenthesis to highlight the complete sequencedivergence of hPDE IV-B2 and hPDE IV-B3 from hPDE IV-B1 at amino acid196.

FIG. 5. Restriction Map of the hPDE IV-B Genomic Locus. Transcriptionalorientation (5′–3′) of hPDE IV-B is from left to right, with theapproximate positions of exons known by partial sequence analysisindicated by solid boxes (coding). The position of the stop codon isindicated by an asterisk, followed by a segment of a 3′ untranslatedregion (open box). Regions hybridizing strongly to the 308 bp probe, asdescribed in the text, are indicated by a dark hatched box, while weaklyhybridizing regions are shown as lighter hatched regions. It is becauseof weak hybridization between the EcoRI and HindIII sites in λ11.1 thatwe position an exon (with a “?”) in that interval. The hybridizingrestriction fragments seen in genomic blots with the 308 bp probe areindicated below the figure.

DETAILED DESCRIPTION OF THE INVENTION

The procedures by which the DNA sequences encoding for novel isozymeshPDE IV-B1, hPDE IV-B2 and hPDE IV-B3 were identified and isolated asdescribed below.

Discovery of PDE IV-B Using Degenerate PCR: The degenerate PCR primers(5′-Deg and 3′-Deg, as described below in the section labelled Materialsand Methods) were designed against amino acid sequences that wereconserved (with one exception) between the six published PDE IVsequences from human, rat, and Drosophila (Uvi GP et al., Mol. Cell.Biol. 10:2678 [1990], Swinnen J V et al., Proc. Nat. Acad. Sci. (USA)86:5325 [1989], and Chen C N et al., Proc. Nat. Acad. Sci. (USA) 83:9313[1986]). These primers were expected to amplify 308 bp of PDE IVsequence from any human isoform mRNA that also conserved those aminoacids. The RT-PCR was done on human BAL sample total RNA as describedbelow in the section labelled Materials and Methods, and a fragment ofthe correct size was obtained. Sequence analysis of this fragment showedit to be different from hPDE IV-A (Uvi GR et al., [1990]). This fragmentof hPDE IV-B corresponds to bp 1,575 to 1,882 in SEQUENCE ID NO. 1. Thisfragment was isolated from several independent PCR reactions andsequenced to confirm that no apparent differences were due to PCRartifacts.

Isolation of a cDNA Clone for hPDE IV-B: The human medulla cDNA librarywas screened as described below in the section labelled in Materials andMethods, and a single cDNA clone was obtained. The insert sequencecorresponds to bp 924 to 2,554 of SEQUENCE ID NO. 1, and was clearly notfull length in the coding region by comparison with the known PDE IVsequences. Also, since no polyA tract was found at the 3′ end of thisclone, we do not believe that the 3′ untranslated region is completehowever, this is of no functional significance with respect to producinga hPDE protein. There was one nucleotide difference between the cDNAsequence and the PCR fragment sequence. SEQUENCE ID NO. 1 contains a Cat bp 1792, the nucleotide seen in the cDNA sequence, rather than the Tthat has been seen at this position in PCR isolations. We believe thatthis difference, which changes an amino acid, is real, and represents analleleic difference in the human population.

Completion of the cDNA Sequence using the RACE Method: The RACE methodshowed that there was not just a single 5′ end to the hPDE IV-B cDNA,but at least three. Fragments of different sizes were obtained, allbeginning at the GSi oligonucleotide primer site and extending towardsthe 5′ end of the cDNA. The three fragments that were successfullysequenced had a variable length of non-homologous sequence at the 5′ endthat joins the hPDE IV sequence at the same point in all three cases.These different 5′ ends, when joined to the rest of the cDNA sequence,make three forms of the hPDE IV-B gene that we designate hPDE IV-B1(SEQUENCE ID NO. 1), hPDE IV-B2 (SEQUENCE ID NO. 2), and hPDE IV-B3(SEQUENCE ID NO. 3). The three hPDE IV-B isoforms make polypeptides ofdifferent lengths. From the cDNA sequences, hPDE IV-B1 is predicted toencode a protein of 721 amino acids (SEQUENCE ID NO. 4), hPDE IV-B2 aprotein of 564 amino acids (SEQUENCE ID NO. 5), and hPDE IV-B3 a proteinof 517 amino acids (SEQUENCE ID NO. 6). The three isoforms are showndiagrammatically in FIG. 1, and the DNA sequence and amino acidtranslation of the three isoforms of hPDE IV-B is shown in FIG. 2.

The most logical explanation for the three hPDE IV-B isoforms is thatthey are generated by alternative splicing of 5′ exons onto the shared3′ sequence. The putative alternative splice junction is shown at −23 bpin FIG. 2. To test this hypothesis, w amplified PCR fragments from humangenomic DNA using primers on either side of the putative splicejunction. hPDE IV-B1 and hPDE IV-B3 specific 5′ primers did not giveamplified fragments, indicating that the sequences on either side of theputative splice lie further than 2 kb apart in genomic DNA (thepractical limit for PCR amplification). Primers specific for the hPDEIV-B2 isoform gave the identically sized fragment as predicted from thecDNA (data not shown), indicating that at least with respect to theputative splice junction at −23 bp, this is the unspliced genomicsequence. Indeed, examination of the sequence of hPDE IV-B2 at thislocation (underlined bp −33 to −24 in FIGS. 2 and 3) reveals anexcellent match for a splice acceptor sequence (Breathnach R and ChambonP, Ann. Rev. Biochem. 50:349 [1981]).

hPDE IV-B is very similar to one of the known rat isozymes, DPD(Colicelli J, et al., Proc. Nat. Acad. Sci. (USA) 86:3599 [1989]), with96.3% amino acid identity in the regions that can be aligned, ascompared to only a 74.6% identity with hPDE IV-A. However, of the threesplice variants, only hPDE IV-B1 continues to have homology to rat DPD5′ of the putative splice junction (FIG. 4). Indeed, hPDE IV-B1 extendsmuch further 5′ than rat DPD, and the homology between the two continuesto the 5′ end of rat DPD. The fact that the hPDE IV-B1 sequence has beenconserved in evolution is strong evidence that this sequence isfunctional and is translated into protein in vivo. We cannot be surethat the other two splice variants are functional in vivo, although therecent paper (McLaughlin MM et al., J. Biol. Chem. 268:6470 [1993])reporting the hPDE IV-B2 sequence has shown by expression cloning thatthis isoform can produce enzymatically active protein in a yeastexpression system.

Mammalian Expression Clones for hPDE IV-B1, -B2, -B3: The hPDE IV-B1,-B2, -B3 cDNA sequences were subcloned into the mammalian expressionvector pcDNA1-amp, a vector that is suitable for transiently expressingthese genes in COS cells and that was constructed by replacing the 950bp NheI fragment of pcDNA1 (Invitrogen) with a 1.2 kb PCR fragment frompUC18 (Sigma) containing the Amp resistance gene. The resultingexpression clones are designated pc-hPDE IV-B1, pc-hPDE IV-B2, andpc-hPDE IV-B3. All three clones have been shown to direct the expressionof proteins that catalyze the degradation of cAMP when transientlytransfected into COS cells.

Genomic Sequences for hPDE IV-B: Overlapping genomic clones define ⁻26kb of genomic sequence encoding at least the 3′ half of the hPDE IV-Bgene (FIG. 5). Limited DNA sequencing of these genomic clones confirmsthat the SalI restriction site in clone λK2.1 is contained in an exon,and corresponds to the unique SalI site seen in the cDNA sequence(1,235–1,240 in Sequence ID No. 1). Hybridization data (FIG. 5) definesthe orientation of the gene, and confirms the hybridizing fragment sizesseen in genomic Southern blots hybridized at high stringency with the308 bp PCR fragment (bp 1,575–1,882 in SECUENCE ID NO. 1) from hPDEIV-B: EcoRI-6.6 kb, HindIII-4.4 kb, BamHI-4.2 kb.

Deposits

Three cDNA clones (pc-hPDE IV-B1, pc-hPDE IV-B2, and pc-hPDE IV-B3) arbeing deposited with the American Type Culture Collection, Rockville,Md. U.S.A. (ATCC).

Assays

Using the DNA sequence of hPDE IV-B and hPDE IV-A, one could make a largnumber of isoenzyme specific PCR primer pairs. We have made and testedthe following hPDE IV-B and hPDE IV-A specific primer pairs. Thesequences 5′B(5′-CGAAGAAAGTTACAAGTTC-3′) and3′B(5′-AACCTGGGATTTTTCCACA-3′) are a pair of 19-mer primers thatspecifically amplify a 245 bp fragment from hPDE IV-8, and the sequences5′A(5′-CACCTGCATCATGTACATG-3′) and 3′A(5′-TCCCGGTTGTCCTCCAAAG-3′) are19-mers that amplify an 850 bp fragment specifically from hPDE IV-A. Inaddition, one skilled in the art could easily design a pair of PCRprimers specific for each of the hPDE IV-B splice variants by using theunique 5′ sequences. Using these primers, one can sensitively assay thepresence of these isozymes in any tissue from which total RNA can beisolated (e.g., by the method of Chomcynski P and N Sacchi, Anal.Biochem. 162:156 1987) by performing an RT-PCR reaction on such totalRNA using the specific primers and then assaying the amount of theappropriately sized DNA PCR product by agarose gel electrophoresis. TheRT-PCR conditions are identical to those described in Materials andMethods, except that the thermocycling parameters are as follows:Denature-94° C., 30 sec; Anneal-55° C. 30 sec; Polymerize-72° C., 60.Amplify for at least 30 cycles.

The claimed DNA sequences of this invention can be reproduced by oneskilled in the art by either PCR amplification of the coding regionusing PCR primers designed from the sequences or by obtaining thedescribed cDNA clones from ATCC directly.

Utility of the Invention

A general utility of the novel human PDE IV genes and their encodedpeptides is to allow screening for human PDE IV isozymespecific/selective drugs that may b improved therapeutics in the areasof asthma and inflammation. The cloned genes make it possible, byexpression cloning methods familiar to those skilled in the art, toproduce active, purified isoenzymes that can be used in PDE IV activityassays (e.g., Davis C W, and Daly J W, J. Cyclic Nucleotide Res. 5:65[1979], Torphy TJ and Cielinski L B, Mol. Pharm. 37:206 [1990]) tomeasure the potency of inhibitors against individual isoenzymes. This istrue both for distinguishing hPDE IV-A and hPDE IV-B selectiv inhibitorsand for distinguishing inhibitors selective between hPDE IV-B1, hPDEIV-B2, or hPDE IV-B3. Since the hPDE IV-B splice variants may each havetheir own tissue distribution and may be pharmacologically separablefrom each other, it may be valuable to screen for inhibitors specificfor individual splice variants.

Genomic sequences are also of utility in the context of drug discovery.It may be valuable to inhibit the mRNA transcription of a particularisoform rather than to inhibit its translated protein. This isparticularly true with hPDE IV-B, since the different splice variantsmay be transcribed from different promoters. There is precedent formultiple promoters directing the transcription of a mouse brain2′,3′-cyclic-nucleotide 3′ phosphodiesterase (Kurihara T et al.,Biochem. Biophys. Res. Comm. 170:1074 [1990]). This invention wouldprovide the means for one skilled in the art to locate multiplepromoters. Isolation of genomic clones containing the promoter(s) andthe 5′-most exons of hPDE IV-B1, hPDE IV-B2, and hPDE IV-B3 may beaccomplished by screening a human genomic library with the unique 5′sequences. Such promoters could then be linked to a convenient reportergene such as firefly luciferase (de Wet J R et al., Mol. Cell. Biol.7:725 [1987]), transfected into a mammalian cell line, and used toscreen for agents that inhibit the activity of the promoter of interestwhile having minimal effect on other promoters.

Another utility of the invention is that the DNA sequences, once known,give the information needed to design assays to specifically detect eachisoenzyme or splice variant. Isozyme-specific PCR primer pairs are butone example of an assay that depends completely on the knowledge of thespecific DNA sequence of the isozyme or splice variant. Such an assayallows detection of mRNA for the isozyme to access the tissuedistribution and biological relevance of each isozyme to a particulardisease state. It also allows identification of cell lines that maynaturally express only one isozyme—a discovery that might obviate theneed to express recombinant genes. If specific hPDE IV isozymes areshown to associated with a particular disease state, the invention wouldbe valuable in the design of diagnostic assays to detect the presence ofisozyme mRNA.

Materials and Methods

(a) Cells/Reagents

Cells from a human bronchoalveolar lavage (BAL) were purchased from theJohns Hopkins University (Dr. M. Liu). Human brainstem tissue waspurchased from the International Institute for the Advancement ofMedicine. Unless noted below, all restriction endonucleases and DNAmodifying enzymes were from Boehringer-Mannheim.

(b) Degenerate RT-PCR

Total RNA was isolated from human tissue as previously described(Chomcynski P and Sacchi N, Anal. Biochem. 162:156 [1987]). To preparean 80 μl reverse transcriptase (RT) reaction, 4 μg total RNA and 4 μgrandom hexamer primers (Pharmacia/LKB) were heated to 90° C. for 5 minin 60 μl RNase free water. After chilling on ice, the reaction wasbrought to 80 μl and the following conditions by the addition ofconcentrated stocks: 1×RT buffer (50 mM Tris pH 8.3, 6 mM magnesiumchloride, 40 mM KCl); 1 mM each dATP, dGTP, dCTP, and dTTP; 1 mMdithiothreitol; 25 U/ml RNasin (Promega); and 900 U/ml AMV reversetranscriptase (RT). Incubate at 42 C for 1 hour, then boil for 5 minutesto inactivate the RT.

A 50 μl PCR reaction was set up by using 3.25 μl of the above reactionmix. Final buffer conditions were (including carryover from RT): 10 mMTris pH 8.3, 50 mM potassium chloride, 1.5 μM magnesium chloride, 10μg/ml bovine serum albumin, 2.5% (v/v) Formamide, 200 μM each dNTP, 0.5pmol/μl each degenerate primer(5′-Deg=5′-CAGGATCCAAPACNATGGTNGAPAC-3′,3′-Deg=5′-GCTCTAGATCNGCCCANGTYTCCCA-3′,where N=A, G, C, or T, P=A or G and Y=C or T) and 0.05 U/μl Amplitaqpolymerase (Perkin Elmer). Amplification was don in a Perkin Elmer 9600PCR thermocycler using the following parameters: denature-94° C., 30sec; anneal-37° C.+0.5° C./cycle, 60 sec+1 sec/cycle; polymerize-72 C,60 sec. Amplify for 35 cycles.

(c) Library Screening

8×10⁵ clones from a commercially available human medulla cDNA library(Clontech # HL 1089a) were screened with an 857 bp DNA fragmentcontaining the entire conserved catalytic domain of hPDE IV-A. Thisfragment was generated by RT-PCR amplification from the Jurkat T-cellline mRNA using unique primers to amplify bp 573–1430 from the PDE IV-Asequence (Uvi GP, et al., Mol. Cell. Bio., 10:2678 [1990]). The fragmentwas labeled to a specific activity >5×10⁸ cpm/μg, and hybridized und rthe following conditions: 6×SSC, 5× Denhardt's Solution (1×Denhardt's=0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serumalbumin), 0.1% sodium dodecyl sulfate (SDS), 100 μg/ml yeast tRNA. Probeconcentration was 4×10⁶ cpm/ml. Filters w r hybridized at 65° C. for >16hours, and then washed to a final stringency of 1×SSC at 55° C.

1×10⁶ clones from a commercially available human genomic library(Clontech #HL1111j) were screened with the 308 bp PCR fragment of hPDEIV-B (bp 1,575 to 1,882 in SEQUENCE ID NO. 1) and the homologousfragment from hPDE IV-A. The screening conditions were as follows:5×SSC, 5× Denhardts solution (see above), 40% formamide, 0.5% sodiumdodecyl sulfate, and 20 μg/ml herring sperm DNA. Probe concentration was4×10⁵ cpm/ml. The filters were hybridized at 42° C. for >16 hours, andthen washed to a final stringency of 0.5×SSC at room temperature. Agenomic library was also constructed in the vector LambdaGEM12 (Promega)using the XhoI half-site method, and 1×10⁶ clones screened under thesame hybridization conditions used for the previous genomic library.

(d) DNA Sequencing

All DNA sequencing was done using an ABI model 373A DNA sequencer on DNAfragments cloned into various pGEM vectors (Promega). Sequencingreactions were done using the Taq sequencing method.

(e) RACE Method

The RACE method (Rapid Amplification of cDNA Ends) was adapted from apublished method (Frohman MA and Martin GR, In: Technicue—a Journal ofMethods in Cell and Molecular Biology, Vol. 1, No. 3, pp. 165–170[1989]). In order to produce the 5′ end of the cDNA, an RT reaction wasperformed on human brainstem total RNA as above with the exception thatthe gene specific RT primer (GS-RT: 5′-GCAAGTTCTGAATTTGT-3′) was at aconcentration of 0.1 pmol/μl. The reaction was incubated at 42° C. for 1hour and then shifted to 52° C. for 30 min. This higher temperatureseems to be critical to avoiding a premature truncation productpresumably caused by a sequence that AMV RT has difficulty readingthrough.

After removing buffers using a Centricon 30 filtration device andconcentrating in a speedvac, one tails the cDNA with dATP using terminaltransferase (TdT) in a 20 μl reaction volume. Final conditions are:1×TdT buffer (40 mM K-Cacodylate pH 6.8, 0.1 mM dithiothreitol), 0.75 mMCoCl₂, 0.2 mM dATP, 1,250 U TdT/ml. Incubate 37° C. for 5 min,inactivate TdT at 65° C. 5 min. This reaction is diluted with water to500 μl and used as a template in a series of nested PCR reactions.

The first PCR amplification (50 ml) uses the same PCR buffer conditionsas above, but uses three primers: the Primer/Adaptor (Ro-dT₁₇:5′-AAGCATCCGTCAGCATCGGCAGGACAAC(T₁₇)-3′) at 0.2 pmol/μl, the ForwardOutside Primer (Ro: 5′-AAGCATCCGTCAGCATC-3′) at 0.5 pmol/μl, and theGene-Specific Reverse Outside Primer (GSo: 5′-ATGGCAGCCAGGATTTC-3′) at0.5 pmol/μl. Taq DNA polymerase is only added after denaturing thereaction to 95° C. for 5 min and equilibrating to 72° C. For the firstcycle, the annealing step is 10 min at 55° C., and the extension is at72° C. for 40 min. After that, cycling parameters (PE 9600 machine) ar:Denature 94° C., 30 sec; Anneal 53° C., 30 sec; Polymerize 72° C., 45sec. Amplify 28 cycles. Dilute this product 20× to serve as template fora second PCR reaction using primers nested just inside those used in thefirst PCR reaction. This greatly increases the specificity of the finalPCR products.

The second 50 μl PCR reaction uses identical buffer conditions to thefirst, and uses 1 μl of the 20× diluted product from the first PCRreaction as template DNA. The primers are the Forward Inside Primer (Ri:AGCATCGGCAGGACAAC-3′) and Gene-Specific Inside Primer (GSi:5′-GGTCGACTGGGCTACAT-3′) both at 0.5 pmol/μl. For 12 cycles, theparameters are the same as the final 28 cycles of the previousamplification. The annealing temperature is then raised to 60° C. foranother 18 cycles. Products are then analyzed on an agarose gel. Theyshould extend from the GSi primer to the 5′ end of the mRNA(s).

Sequence ID Summary

-   1. hPDE IV-B1 cDNA sequence. 2,554 bp.-   2. hPDE IV-B2 cDNA sequence. 2,246 bp.-   3. hPDE IV-B3 cDNA sequence. 2,045 bp.-   4. Predicted amino acid sequence of hPDE IV-B1. 721 amino acids.-   5. Predicted amino acid sequence of hPDE IV-B2. 564 amino acids.-   6. Predicted amino acid sequence of hPDE IV-B3. 517 amino acids.

1. An isolated polypeptide comprising the amino acid sequence of one ofSEQUENCE ID NO:4, SEQUENCE ID NO:5 or SEQUENCE ID NO:6.
 2. A polypeptideproduced by expression in a host cell into which has been incorporatedthe DNA sequence of: (a) SEQUENCE ID NO:1 (b) SEQUENCE ID NO:2 or (c)SEQUENCE ID NO:3.
 3. An isolated polypeptide of claim 1 comprising theamino acid sequence of SEQ ID NO: b
 4. 4. An isolated polypeptide ofclaim 1 comprising the amino acid sequence of SEQ ID NO:5.
 5. Anisolated polypeptide of claim 1 comprising the amino acid sequence ofSEQ ID NO:6.
 6. A recombinant phosphodiesterase encoding a polypeptide,said polypeptide comprising; the amino acid sequence of one of SEQ IDNO:4, SEQ ID NO:5 or SEQ ID NO:6.
 7. A polypeptide according to claim 3comprising the amino acid sequence of SEQ ID NO:4.
 8. A polypeptideaccording to claim 6 comprising the amino acid sequence of SEQ ID NO:5.9. A polypeptide according to claim 6 comprising the amino acid sequenceof SEQ ID NO:6.
 10. An isolated polypeptide comprising: (a) an aminoacid sequence of one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and; (b)an amino acid sequence having at least 96.3% sequence identity to one ofSEQ ID NO:4, and wherein said amino acid sequence has phosphodiesteraseactivity.